Many biochemical procedures require isolating cells of a uniform type from a tissue containing a mixture of cell types. Flow cytometry is a technology that can simultaneously measure and then analyze physical characteristics of single particles, usually cells, as they flow in a fluid stream through a beam of light. For example, cell separation is often used to analyze the DNA content of individual cells. In a typical cell-separation technique, DNA or antibodies coupled to a fluorescent dye are used to label specific cells. FIG. 1 is a schematic illustration of a typical flow cytometer, wherein the individual cells, traveling single file, pass through a laser beam and the fluorescence of each cell is measured. For optimal illumination, the cells should be positioned in the center of the laser beam and only one cell should move through the laser beam at a given moment. Labeled cells can then be separated from unlabeled cells, for example using a vibrating nozzle to form droplets containing single cells. This technique can sort many thousands of cells per second.
However, commercial flow cytometers are bulky and expensive, precluding their use in field clinics, water monitoring, agriculture/veterinary diagnostics, and rapidly deployable biothreat detection. Much of the size and cost of conventional cytometers is dictated by the high speed achieved by cells or beads in a hydrodynamically focused stream. Recently, flow cytometry systems based on microfluidics have promised less expensive and portable alternatives to conventional systems. See R. Yang et al., Sensors and Actuators A 118, 259 (2005); and C. Simonnet and A. Groisman, Analytical Chemistry 78, 5653 (2006). These flow cytometry microsystems take advantage of the ability of micromachining technology to pattern small features and integrate multiple sensing modalities (optical, electrical, mechanical) onto a single platform. In addition, microfluidics offers the ability to build complex interrogation channels that can focus cells into narrow single-file columns for downstream optical interrogation. However, underlying issues (e.g., cell clumping and adhesion to the channel) and appropriate routing of the particles to necessary points in an integrated system remain problematic.
Acoustic force manipulation technologies have matured in the last ten years to enable reliable handling of particles in microsystems. See W. T. Coakley et al., Ultrasonics 38, 638 (2000); J. J. Hawkes and W. T. Coakley, Sensors and Actuators B 75, 213 (2001); A. Haake and J. Dual, Ultrasonics 40, 317 (2002); N. R. Harris et al., Sensors and Actuators B 95, 425 (2003); and A. Nilsson et al., Lab on a Chip 4, 131 (2004). These long-range acoustic forces can span the entire dimensions of large (e.g., hundreds of microns) fluidic channels. Moreover, the acoustic forces can be spatially decoupled to strengthen primary radiation forces in one dimension and reduce those in other directions. Also, the acoustic radiation forces that move particles from one position to another act on a different spatial scale from weaker interparticle forces. However, there are certain drawbacks to the use of acoustic forces alone to manipulate particles. For instance, the fabrication of the acoustic device requires non-traditional transducer materials that are difficult to integrate into microsystems. Further, acoustic focusing parameters can generally be changed only by changing frequencies to modify the number of standing wave nodes in the channel. Finally, coupling acoustic forces to devices is not straightforward and poses microfabrication limitations.
Meanwhile, dielectrophoresis (DEP) has been used for many applications in microsystems during the past few years. The DEP force enables particles to be moved precisely from one location to another using simple metal electrode configurations. Negative DEP has been used to levitate single particles at a particular point in a microsystem and to manipulate small groups of particles. See D. Holmes et al., IEE Proceedings-Nanobiotechnology 152, 129 (2005); J. H. Nieuwenhuis et al., IEEE Sensors Journal 5, 810 (2005); and D. Holmes et al., Biosensors and Bioelectronics 21, 1621 (2006). In many of these applications, the particles are manipulated for downstream analyses, such as impedance spectroscopy or chemical lysing. In many ways, DEP forces are complementary to acoustic forces. For instance, DEP forces are flexible and can be modified simply by changing frequencies or by addressing different sets of electrodes in an array to modify the shape of the DEP focusing.
Recently, Wiklund et al. have combined long-range ultrasonic standing wave (USWs), suitable for high-throughput manipulation of multi-particle aggregates, with short-range DEP, suitable for well-controlled and precise handling of individual cells, in a microfluidic chip. See M. Wiklund et al., Lab on a Chip 6, 1537 (2006), which is incorporated herein by reference. By combining USW and DEP, acoustic forces were used to accumulate particles into the multi-particle aggregates, and DEP forces were used to switch and combine particles between adjacent pressure nodes of the USWs in a microfluidic channel. However, Wiklund et al. used an acoustic transducer placed obliquely on a microfluidic chip to achieve lateral focusing of the bioparticles along the length of a microfluidic channel. With oblique coupling, the incident acoustic wave was transferred from a primarily vertical direction to a primarily horizontal direction by carefully matching of the transducer angle and the acoustic properties of the layers between the transducer and the horizontal channel, resulting in a complicated microsystem design.
Therefore, a need remains for a simplified microfabricated particle focusing device that enables the combination of acoustic focusing for large-scale particle preconcentration and dielectrophoretic forces for single-particle focusing and sorting of the preconcentrated particles.